Biomedical Sciences ETDs

Shujie Yang

Publication Date

5-1-2009

Abstract

The ErbB family of receptor tyrosine kinases plays an important role in the development and pathogenesis of many cancers and two of its members, ErbB1 and ErbB2, are already important cancer therapeutic targets. The first goal of this project was to evaluate novel aspects of ErbB signaling complexity, by studying fine-scale membrane organization of three ErbB receptors (ErbB1, ErbB2 and ErbB3) under conditions of receptor overexpression and ligand-induced signaling in breast cancer cells. We used innovative immunoelectron microscopy techniques to map the relative proximity of ErbB family members on SKBR3 breast cancer cell membrane sheets, before and after introduction of ligands. Based upon statistical evaluation of TEM images acquired from double-labeled membrane sheets, we demonstrate that ErbB receptors are predominantly involved in homo-interactions, with markedly less heterodimerization than predicted by previous studies. A second focus was ErbB3, the ErbB family member with impaired kinase activity, that has recently been implicated in escape from ErbB-targeted tumor therapies. Our work suggests that at least two mechanisms amplify ErbB3 signaling and render it partially independent of its preferred heterodimerizing partner, ErbB2. First, robust tyrosine kinase activity in ErbB3 immunoprecipitates from Heregulin-stimulated SKBR3 cells suggests that ErbB3 may compensate for its poor catalytic capabilities by association with an unknown cytoplasmic tyrosine kinase. Second, we discovered that SKBR3 cells express two ErbB3 alleles, the wild type form and a form with a single kinase domain substitution (E933Q). We found ErbB3 activation in SKBR3 cells, and in transfected CHO cells, is only partially blocked by the therapeutic antibody 2C4 (Pertuzumab), that blocks heterodimerization with ErbB2. Remarkably, the E933Q substitution increases ligand binding affinity and amplifies the phosphoinositol 3-kinase (PI3K)/AKT cell survival/ mitogenic pathway. These results suggest that multiple mechanisms contribute to ErbB3s critical roles in tumor survival and, further, that ErbB3 and its cytosolic partners are potential therapeutic targets. These studies have revealed novel aspects of ErbB receptor membrane organization during signaling and provide the first description of an ErbB3 gain-of-function mutation. This work advances the understanding of the molecular, structural and biological characteristics of the ErbB family and offers insight into mechanisms underlying ErbB-targeted therapies.

Keywords

ErbB receptor, signal transduction, membrane topography, breast cancer, ErbB3 mutation

Sponsors

National Institutes of Health and Oxnard Foundation

Document Type

Dissertation

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Doctoral

Department Name

Biomedical Sciences Graduate Program

First Committee Member (Chair)

Oliver, Janet

Second Committee Member

Lidke, Diane

Third Committee Member

Leslie, Kimberly

Fourth Committee Member

Edwards, Jeremy

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